Generator

Part:BBa_K1499004:Experience

Designed by: Raman Nelakanti   Group: iGEM14_StanfordBrownSpelman   (2014-10-08)
Revision as of 00:24, 20 October 2016 by MattBorel (Talk | contribs)


This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K1499004

Group: 2016 Stanford-Brown iGEM Team Author: Michael Becich Summary: The 2016 Stanford-Brown iGEM Team purified this linker protein and used it to create a BioDevice. Used in tandem with a biotinylated fluorophore, this CBD/Streptavidin fusion protein served as a linker between cellulose paper and the fluorophore-quencher biosensor described here: http://2016.igem.org/Team:Stanford-Brown/SB16_BioSensor_FQsensor. Uploads: (links to uploads relevant to your contribution, ex: csv containing your data, sequence files, etc.)

Group: 2016 INSA-Lyon iGEM Team

Author: Mathieu Borel

Summary: The 2016 INSA-Lyon iGEM Team purified and characterized this part. The team showed it was possible to purify this part using affinity chromatography on a cellulose column. With a biotinylated and Fluorescent labelled DNA oligo the team also showed it was able to bind at the same time cellulose and biotin. You can see our proof of concept page for further details on how we used this part in our system: http://2016.igem.org/Team:INSA-Lyon/Proof

User Reviews

UNIQf27d707f8d1290e9-partinfo-00000000-QINU

••••

mbecich

The part worked as specified for our needs! It should be noted that an inducible promoter, HisTag, and double terminator are all included in this part to facilitate expression and purification. This was confirmed to us by the part creator and former Stanford-Brown iGEMer herself, Alaina Shumate.

BBa_K1499004 1 Not understood INSA-Lyon

INSA Lyon 2016 Experiments on this part

This part contains the sequence coding for the streptavidin protein linked to two cellulose-binding domains, one located at the N-terminus (CBD1 - BBa_K1934080) and one located at the C-terminus (CBD2 - BBa_K1934090) of the protein sequence.

Streptavidin is a 52.8 kDa protein which has the ability to bind to biotin with high affinity. Indeed, this can be explained by its structure and the formation of an extensive network of intramolecular interactions when biotin is in the binding site. This strong noncovalent link is used in many biotechnologies especially in purification and detection assays. The combination of streptavidin with other proteins moreover enables to confer new properties such as a cellulose-binding activity with the integration of two CBDs [1]. The part BBa_K1499004, cellulose binding domains with streptavidin domain generator, was submitted to the iGEM registry in 2014, but was missing transcriptional and translational signals, and experimental data. Therefore, the part BBa_K1934020 was designed as a complete streptavidin-CBDs generator displaying a promoter, RBS and terminator.

[1] Bayer, E. A., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and cellulosomes. Current opinion in structural biology, 8(5), 548-557.

Characterization

Purification Using Cellulose Affinity

The BBa_K1934020 part conceived by the 2016 INSA-Lyon team and synthesized by IDT was cloned into pSB1C3 and transformed into the E. coli NM522 strain. One recombinant clone was grown overnight in LB at 24°C, with IPTG 1 mmol/L-1 and glucose 5 mmol/L-1. Cells were harvested and resuspended in 1 mL lysis buffer (50 mmol/L-1 Tris, 300 mmol/L-1 NaCl, 10% glycerol). Then the mix was sonicated 5 times 30 seconds on ice at moderate power. The lysate was centrifuged at 14,000 g for 10 min. The supernatant was treated as follow:

  • Wash microcrystaline Cellulose five times in water. Then equilibrate in washing buffer (ammonium sulfate 1M). Pack the cellulose (10x10mm) in small chromatography columns (we used syringes barrels).
  • Gently pour the lysate supernatant on the column. Once the liquid starts flowing through evenly, measure the OD280 of the different fractions. Continue pouring washing buffer until the OD280 stabilizes around zero.
  • Change the washing buffer to water. OD280 shortly rises. Keep the fractions with the highest OD280 . They should contain the protein.
  • Analyse collected fractions on an SDS-PAGE. Optionally, proteins may be concentrated using ultrafiltration.

Figure 1. Purification of the chimeric Streptavidin-CBD protein on a cellulose column This elution graph shows a first peak, present for both the control and our expression culture. This first peak corresponds to unbound proteins. In the presence of water, only one peak was observed: it’s the elution peak of our protein.

2. BBa_K1934020 encodes a protein able to bind both biotin and cellulose

Affinity of the streptavidin-CBD encoded by BBa_K1934020 to cellulose was compared to the one of commercial streptavidin. A molecule of fluorescein was grafted at the 5’ end of a DNA oligo carrying a molecule of biotin at its 3’ end. This DNA oligo constitutes the reporter system. Such modified oligo was mixed either with the engineered streptavidin-CBD or with commercial streptavidin. The resulting mix was incubated with microcrystalline cellulose in presence of PBS for 1 hour. The cellulose was then washed twice with fresh PBS and fluorescence was measured. Every experiment was done in triplicate.

Figure 2. The Streptavidin-CBD is able to bind biotin and cellulose. Mixed raw cellulose with our report system shows no fluorescence (first bar). The measured fluorescence indicates that commercial streptavidin was able to bind our reporter system and sticks at a low extent to cellulose. We concluded that this results from none-specific adsorption. For the streptavidin-CBD part (BBa_K1934020), a high fluorescent signal was recorded.
This experiment shows that this streptavidin-CBD protein is able to bind efficiently biotin and cellulose at the same time. The same experiment was done for the [[Part:BBa_K1934030|BBa_K1934030]]: part displaying a different cellulose binding domain, namely CBD-CipA. The binding efficiency of streptavidin-CBDs tends to be slightly lower compared to streptavidin-CipA (x1.1) but was not statistically demonstrated.

••••

mbecich

The part worked as specified for our needs! We cloned it in pSB1C3, with a pTac promotor, strong RBS and a double terminator. It is important to note that this protein tends to dimerize when it is over-expressed driving to a loss of function.


UNIQf27d707f8d1290e9-partinfo-00000005-QINU